Biomedical subjects

W G Breed

Publications and source records attributed to W G Breed.

At least 55 records · Page 3Linked to original sources

PubMed1994

Microtubule configurations in oocytes, zygotes, and early embryos of a marsupial, Monodelphis domestica.

The marsupials represent a separate evolutionary lineage from eutherians from which they diverged over 100 million years ago. In order to explore the origin and mode of centrosome inheritance amongst this group of mammals, this study investigates the microtubule organization during fertilization, parthenogenesis, and polyspermy in the didelphid, Monodelphis domestica. Microtubules and DNA were visualized in maturing ovarian oocytes, parthenogenetically activated oocytes, monospermic and polyspermic zygotes, and early embryos. Ovarian oocytes had a central region of yolky cytoplasm that, after fertilization, became polarized; much of the yolk was then extruded into the perivitelline space as an enucleated cytoplasmic mass. Immunofluorescence microscopy, using a monoclonal antibody to beta-tubulin, demonstrated microtubules in the meiotic spindle in unfertilized oocytes, but cytasters were not detected. After fertilization, a cluster of microtubules forming into a sperm aster was evident around the male pronucleus. The sperm aster remained largely restricted to the nonyolky region of the egg cytoplasm, resulting in a cytoplasmic heterogeneity between a microtubule-rich region and one in which microtubules were largely absent. Once the two pronuclei came close together, abundant microtubules were found surrounding both pronuclei. In the early embryo, microtubules were found in the outer cortical region of the blastomeres and, in addition, there was an extensive and elaborate network of microtubules throughout the yolk mass. Disruption of the meiotic spindle microtubules with nocodazole or cold treatment did not result in chromosome dispersion in the cortex and recovery from drug or cold depolymerization demonstrated that microtubules might not be as dynamic as those in eutherian mammals. Taxol stabilization resulted in an increase in cortical microtubules. In this marsupial species, therefore, the centrosome appears to be of paternal origin, and the radiating microtubules that form may well be involved both in bringing the pronuclei together and in the cytoplasmic polarization that results in extrusion of the yolk mass.

Animals↗

PubMed1994

Sperm-egg interactions in an Australian dasyurid marsupial with official reference to changes in acrosomal morphology.

The morphology of the acrosome of spermatozoa from an Australian dasyurid marsupial during in vivo fertilisation, as seen with the transmission electron microscope, is described. In some spermatozoa close to the outer zona surface with largely intact acrosomes, electron-dense struts between the inner and outer acrosomal membranes could be seen; in addition fusion between the outer acrosomal and overlying plasma membrane was sometimes evident. The zona matrix close to the acrosomal region of spermatozoa with heads partly embedded within the zona pellucida was less electron-dense and more filamentous than elsewhere; perhaps indicative of local lytic activity. Nevertheless some of these spermatozoa at least had partly intact acrosomes with localised regions of electron-dense bridges between the two acrosomal membranes. Spermatozoa with condensed chromatin were also observed within the egg cytoplasm some of which had membranes, vacuoles or cisternae close to the dorsal nuclear surface which could, in part, be acrosomal in origin. These observations lend some support to the recent view, based on findings from an American species, that acrosomal changes of spermatozoa of some marsupials at the time of fertilisation may be somewhat similar to those of eutherian mammals in spite of the considerable differences in gamete structure and design. How generally applicable these findings are to all marsupials remains, however, to be determined.

Acrosome↗

PubMed1994

How does sperm meet egg?--in a marsupial.

Australian marsupials exhibit a wide range of variation in sperm head morphology, and in thickness of the zona pellucida around the oocyte, suggesting interspecfic differences in the processes of sperm-egg interaction. The observations described here are largely based on the dasyurid Sminthopsis crassicaudata. They show that in oestrous females, after mating, a coagulum forms in the lateral vaginae and, within an hour of insemination, numerous spermatozoa congregate in the isthmus of the oviduct in which the vanguard population undergoes transformation with the head rotating on its axis with the tail to form a T-shape. Once oocytes are released, a few spermatozoa migrate to the higher reaches of the oviduct where sperm-zona binding occurs by way of the plasmalemma over the acrosomal region. The acrosome reaction takes place here and, as the egg rotates, the tail of the spermatozoon becomes parallel to the head. A small region of acrosome sometimes appears to remain intact at this time because spermatozoa with partly intact acrosomes have been found within the zona matrix. In some of these, electron-dense bridges between part of the inner and outer acrosomal membranes which may act as stabilizing structures, were also seen. The zona matrix is tightly packed around the penetrating spermatozoon, but that close to the acrosomal region becomes less electron-dense and more filamentous. Once incorporated into the egg, the spermatozoon lacks a cell membrane around the tail but vesicles close to the sperm head may, at least in part, be remnants of an inner acrosomal membrane. How generally applicable these observations are to other Australian marsupials remains to be determined.

Animals↗

PubMed1994

Regulated storage and subsequent transformation of spermatozoa in the fallopian tubes of an Australian marsupial, Sminthopsis crassicaudata.

The disposition of spermatozoa in the female tract of the dasyurid marsupial, Sminthopsis crassicaudata, was examined before and after ovulation, by transillumination using differential interference optics. At 6-30 h after mating in females with maturing follicles, spermatozoa were restricted to special storage crypts that define the isthmus of the Fallopian tube. These spermatozoa were mostly immotile and were spear shaped, except for 50-200 vanguard spermatozoa within anterior crypts that were T shaped due to a rotation of the head on the tail. After ovulation and arrival of eggs in the narrow upper ovarian segment of the tube, several hundred isthmic spermatozoa migrated to that region, dispersed singly or in small groups of variable motility with some festooned around eggs. Most clearly displayed a T configuration, and subsequently, those remaining in the isthmic crypts also adopted a T shape in situ. The motility of mature epididymal spermatozoa and of those flushed from the isthmus before and after ovulation was similar in vitro, and all were spear shaped except for some T-shaped isthmic spermatozoa recovered after ovulation. However, neither spear- nor T-shaped active isthmic spermatozoa ever adhered to the zona pellucida when incubated with eggs. We conclude that transport to and storage of spermatozoa in the isthmus of the Fallopian tube, and their later migration up to the fertilization site and coincident transformation to a T shape, all are closely regulated by the ovarian follicular cycle in Sminthopsis. Only on reaching the upper ovarian segment of the Fallopian tube do Sminthopsis spermatozoa appear to be able to bind to the egg, when the T shape brings the whole surface area of the acrosome into apposition with the zona pellucida. These observations are discussed with special reference to sperm capacitation, sperm penetration mechanisms, and the different idiosyncratic designs of the sperm head in marsupial and eutherian mammals, respectively.

Animals↗

PubMed1994

Placentation in the dasyurid marsupial, Sminthopsis crassicaudata, the fat-tailed dunnart, and notes on placentation of the didelphid, Monodelphis domestica.

A light microscope study of the choriovitelline (yolk sac) placenta of the dasyurid marsupial, Sminthopsis crassicaudata, and some comparative observations on that of the didelphid, Monodelphis domestica, were performed. In the former species, the placenta was composed of an invasive bilaminar, avascular, yolk sac and a non-invasive trilaminar, vascular yolk sac. The bilaminar yolk sac placenta had trophoblast giant cells that eroded the maternal epithelium, but there was no evidence of invasion of maternal capillaries; thus, an endotheliochorial placenta was present. In the trilaminar yolk sac placenta, the convoluted chorion followed the contours of the highly folded endometrial epithelium but did not erode it and, therefore, an epitheliochorial placenta was formed. In late pregnancy, the choriovitelline placenta of Monodelphis domestica also had two regions, but the fetal trophoblast did not invade the uterine epithelium in either region. Rather, there were discontinuous areas of adhesion between trophoblast giant cells and uterine epithelium in the trilaminar yolk sac placenta and some extensive areas of adhesion in the attenuated bilaminar yolk sac placenta. The yolk sac placenta in M. domestica, unlike that of S. crassicaudata, therefore appears to be epitheliochorial in the vascular and non-vascular regions.

Animals↗

PubMed1994

Effect of cryopreservation on development and ultrastructure of preimplantation embryos from the dasyurid marsupial Sminthopsis crassicaudata.

The effect of different cryopreservation methods on the development and ultrastructure of preimplantation embryos of Sminthopsis crassicaudata, a small carnivorous marsupial and member of the family Dasyuridae, was investigated. Females were primed with 1 iu pregnant mares' serum gonadotrophin to induce oestrus and ovulation. Mating generally ensued and, approximately 6 days after priming, embryos were collected and cultured in 5% CO2 in air at 35 degrees C for 18-22 h in either Dulbecco's modified Eagles medium (DMEM) with high glucose or human tubal fluid medium (HTF), both supplemented with 10% fetal calf serum. Cleavage rates were higher in DMEM than in HTF. One slow and two ultrarapid freezing methods were used. Two out of 12 (17%) embryos cleaved in culture after freezing and thawing using the slow regimen, compared with six of 16 (38%) non-frozen controls. In addition, two of 11 (18%) embryos cleaved in culture following ultrarapid freezing and thawing by one of the two methods, compared to 31 of 41 (76%) non-frozen controls. Most of the embryos appeared morphologically normal under the light microscope after freezing and thawing by the slow regimen, but considerable variation in the degree of ultrastructural damage to the cellular organelles was evident with the transmission electron microscope. The rather low rate of cleavage after freezing and thawing was probably due, at least in part, to ultrastructural damage of the cells.

Animals↗

PubMed1993

In vivo parthenogenetic activation of ovulated oocytes in a marsupial, Sminthopsis crassicaudata.

The occurrence of in vivo parthenogenesis is documented for laboratory-bred individuals of the Australian dasyurid marsupial, Sminthopsis crassicaudata. About 30% of females that had been isolated from males for a greater period of time than the length of pregnancy were found, on dissection of their uteri, to have embryos present. The embryos were surrounded by a mucoid coat and shell membrane and at least the first two cleavage divisions occurred normally. After this time, however, unequal cleavage divisions appeared to result. The parthenogenesis that takes place may be initiated as a result of activation of ageing ovulated oocytes. Its frequent occurrence may prove useful in a study of maternal and paternal contributions to early embryonic development in the species.

Animals↗

PubMed1993

Novel organization of the spermatozoon in two species of murid rodents from southern Asia.

The morphology and structural organization of unusual spermatozoa from two species of bandicoot rats (Bandicota spp) from Southern Asia were studied with the light and transmission electron microscope. Unlike those described for nearly all other eutherian species the heads of Bandicota savilei and B. indica spermatozoa were bulbous or globular in shape and had no narrow uniformly defined apex nor a clearly delineated perforatorium. The nucleus, which had one or more prominent vacuoles, was capped by a huge, bulky, acrosome with no obvious morphologically recognizable equatorial segment. The postacrosomal dense lamina was extremely short and a posterior ring lay anterior to the caudal pole of the nucleus. In addition, the tail of the spermatozoon was only 28-45 microns in length. The organization of the spermatozoon of these two species of Bandicota is thus quite unlike that described for any other murid rodent or even mammalian species. Its highly divergent features suggest that the processes of zona penetration and incorporation of the fertilizing spermatozoon into the egg may differ markedly from those of all eutherians so far investigated.

Acrosome↗

PubMed1992

Marsupial fertilization: some further ultrastructural observations on the dasyurid Sminthopsis crassicaudata.

Various morphological aspects of in vivo egg maturation and sperm-egg interaction were investigated in the Australian marsupial Sminthopsis crassicaudata with the transmission and scanning electron microscopes. Cortical granules invariably occurred in primary oocytes, with the number increasing after resumption of the first meiotic division. They generally occurred close to the oolemma, including the region near the oocyte nucleus. After mating, spermatozoa with intact acrosomes, which had a homogeneous electron-dense matrix, were found on the outer zona surface, but loss of acrosomal contents had occurred by the time of zona penetration. Sperm incorporation into the egg took place at the metaphase II stage of meiosis, and, at this time, cortical granules disappeared from the egg cortex. Sperm heads with condensed chromatin in the egg cytoplasm had an electron-dense layer of subacrosomal material over part of the dorsal nuclear surface, but no membranes were present around these incorporated spermatozoa. Sperm chromatin decondensation resulted in an elevation of egg cytoplasm, and the cell membrane over this area lacked microvilli. The pronuclear envelope was not laid down until after chromatin decondensation had occurred. By this time the fertilized egg had reached the uterus, and a smooth, electron-dense, shell membrane had been deposited. These observations, together with our previous findings, indicate that some of the processes of sperm-egg interaction are similar to those in eutherian mammals, whereas others appear highly divergent.

Animals↗

PubMed1992

Gonadotrophin-induced oestrus and ovulation in the polyovulatory marsupial Sminthopsis crassicaudata.

Sminthopsis crassicaudata is a small (approximately 16 g) polyovulatory dasyurid marsupial which has the potential to become an important model species. This study examined the use of exogenous hormone treatment to manipulate the breeding of S. crassicaudata and as a means to obtain timed developmental stages for further study. Two thirds (21/32) of the females treated with 1.0 or 5.0 I.U. of pregnant mare serum gonadotrophin (PMSG) had ovulated when the contents of their reproductive tracts were examined 5 or 6 days later. Only one of eight females treated with 0.2 I.U. PMSG had ovulated in the same period. Although a similar proportion of animals treated with 1.0 I.U. and 5.0 I.U. ovulated, the ovulation rate was significantly lower when the higher dose was administered (mean of 10.5 ovulations per female v. 3.8 ovulations per female). In addition, the ovaries of 6/8 of the animals treated with 5.0 I.U. PMSG had luteinized follicles with degenerating oocytes, evidence of over-stimulation. Follicular luteinization also occurred in 4/8 animals treated with 1 I.U. PMSG. Oocyte maturation and ovulation occurred following PMSG stimulation without injection of synthetic gonadotrophin releasing hormone (GnRH). Treatment with a 10-micrograms dose of GnRH following PMSG seemed to have no effect on the outcome. Of the females that had ovulated by Day 6, three quarters had mated and some had fertilized eggs and two-cell embryos in the oviducts and uteri. In a further series of experiments the subsequent development of embryos conceived after PMSG treatment was assessed. Two thirds of treated females mated within 7 days of treatment and 60% of these matings yielded embryos when examined 11 days after PMSG. However, full-term development was only achieved in one animal. Gonadotrophin treatment of S. crassicaudata thus may have application as a means to obtain mature or maturing oocytes, cleavage stage embryos and blastocysts, but at this stage it appears not to offer promise as a method to achieve full-term development.

Animal Husbandry↗

PubMed1991

Distribution of filamentous actin in and around spermatids and in spermatozoa of Australian conilurine rodents.

The distribution of filamentous actin around the maturing sperm head and in spermatozoa of four species of Australian conilurine rodents was investigated at the light and electron microscopic levels. Similar results were obtained for all the species studied. Mechanically isolated spermatids had NBD-phallacidin-positive longitudinal bands of fluorescence over the dorsolateral surface and, in late spermatids, bands of bright fluorescence passed perpendicularly from the dorsal convex to ventral concave surface. TEM observations indicated that these regions corresponded to filaments of ectoplasmic specializations and granular filamentous material around the tubulobulbar complexes, respectively. In testicular and cauda spermatozoa NBD-phallacidin fluorescent material was present in the two ventral processes that extended from the upper concave surface of the sperm head; also fainter material occurred along the concave border and as a dorsocaudal spur. Its distribution was identical for testicular and cauda spermatozoa. TEM of late spermatids showed that in the ventral process closest to the apical hook there were between 170 and 245 filaments, which attached to the inner surface of the postacrosomal dense lamina; in the more caudal ventral process about 70 filaments occurred. No filaments were, however, visible in the mature spermatozoon but, after immunocytochemical labelling for actin, deposition of gold particles was evident over ventral processes of both late spermatids and cauda spermatozoa. Within the female tract these ventral processes made contact with the zona matrix and were taken into the egg cytoplasm unchanged in morphology. The possible functional significance of the filamentous actin in these structures is discussed.

Acrosome↗

PubMed1991

Transillumination patterns of seminiferous tubules in two species of Australian rodents, Pseudomys australis and Notomys alexis.

The transillumination-assisted isolation of segments of seminiferous tubules in defined stages of the wave of the seminiferous epithelium was investigated in Pseudomys australis and Notomys alexis. In P. australis, three different transillumination patterns (pale, spotty and dark) of the seminiferous tubules could be isolated. They corresponded to histological stages 1-2, 3-5 and 6-8 of the wave of the seminiferous epithelium. The dark pattern made up about 40% of the wave, the pale pattern about 31%, and the spotty pattern about 24%. In N. alexis, no such isolation of segments with particular cell associations was possible. In a comparison of P. australis with the laboratory rat, the dark, pale and spotty transillumination patterns of seminiferous tubule segments corresponded to the same cell associations in both species, but the length of the spotty segments in the laboratory rat made it possible to subdivide the spotty region into pale spotty and dark spotty areas and thus to isolate four different transillumination patterns. Further separation into other subgroups to isolate additional types of segments could not be performed repeatedly in any of the studied species because of the gradual change of one subgroup of transillumination pattern into another and the short length of some of the subgroups.

Animals↗

PubMed1991

Mating behaviour and insemination in the hopping mouse (Notomys alexis).

Hopping mice have extremely small testes and a reduced complement of male accessory sex glands. Nevertheless, greater than 1000 spermatozoa populate the isthmus of the oviduct after each insemination and a range of morphological types is found similar to that in the male tract. When females are primed with gonadotrophins they sometimes lock with more than one of the males within the group and in the present study two inseminations sometimes took place at the one oestrus. There is thus the possibility for intermale sperm competition within the female tract of these animals.

Animals↗

PubMed1990

Morphological changes in the oocyte and its surrounding vestments during in vivo fertilization in the dasyurid marsupial Sminthopsis crassicaudata.

This light and transmission electron microscopical study shows that the first polar body is given off before ovulation and that part of its cell membrane and that of the surrounding oocyte have long microvilli at the time of its ejection. Several layers of cumulus cells initially surround the secondary oocyte and first polar body, but the ovulated oocytes in the oviducts in the process of being fertilized do not have cumulus cells around them. Partly expelled second polar bodies occur in the oviduct; they are elongated structures that lack organelles and have electron-dense nuclei. A small fertilization cone appears to form around the sperm tail at the time of sperm entry into the egg and an incorporation cone develops around the sperm head in the egg cytoplasm. In three fertilized eggs a small hole was seen in the zona, which was presumably formed by the spermatozoon during penetration. Cortical granules, present in ovarian oocytes, are not seen in fertilized tubal or uterine eggs; release of their contents probably reduces the chances of polyspermy, although at least one polyspermic fertilized egg was seen and several other fertilized eggs had spermatozoa within the zona pellucida. In the zygote the pronuclei come to lie close together, but there was no evidence of fusion. A "yolk mass," which becomes eccentric before ovulation, is extruded by the time the two-cell embryos are formed, but many vacuoles remain in the non-yolky pole of the egg. A shell membrane of variable thickness is present around all uterine eggs but its origin remains undetermined.

Animals↗

PubMed1990

Organization of testicular interstitial tissue of an Australian rodent, the spinifex hopping mouse, Notomys alexis.

The organization of testicular interstitial tissue of the spinifex hopping mouse, Notomys alexis differs from that of other rodents. It comprises between 10.3% and 17.3% (average 15.0%) of the total testicular volume, and is variable in its organization both at different locations within the testis of the one animal and among different individuals. Abundant, closely packed Leydig cells are usually present; however, in some regions large, thick-walled blood vessels and extensive peritubular lymphatic spaces, often lacking an endothelium adjacent to the Leydig cells, are also prominent. The Leydig cells in contact with the large blood vessels and lymphatics, unlike those in regions where lymph is sparse, are not densely packed and sometimes contain numerous lipid droplets. Ultrastructure of Leydig cells is typical of steroid-producing cells; however, mitochondria are often extremely large, unusual in shape or bizarrely arranged in relation to one another. Also electron-dense bodies displaying a paracrystalline-like internal structure of parallel, electron-dense filaments arranged in a lattice pattern occur in the cytoplasm of many cells. The significance of these unusual ultrastructural features and the organization of the interstitial tissue remain to be determined conclusively, but may relate to steroid synthesis, secretion and uptake.

Animals↗

PubMed1990

Copulatory behaviour and coagulum formation in the female reproductive tract of the Australian hopping mouse, Notomys alexis.

Hopping mice were examined to study two interrelated questions: (1) when groups of adults of both sexes are kept together in one cage in the laboratory is there evidence that the females copulate with only one male, and (2) is a copulatory plug formed in the female tract after ejaculation? The findings indicate that a female will sometimes lock with more than one male in the group during an oestrous period induced by administration of exogenous gonadotrophins, and that a small 'plug' of soft material forms post coitum in the more caudal parts of the female tract. Individuals of this species, therefore, do not appear to be strictly monogamous, at least in this artificial laboratory situation. Although a coagulum is formed, this is quite different from the typical hard copulatory plug that occurs in common laboratory murids; it may possibly reduce sperm backflow from the lower region of the female reproductive tract.

Animals↗

PubMed1989

Sperm morphology and storage in the female reproductive tract of the fat-tailed dunnart, Sminthopsis crassicaudata (Marsupialia: Dasyuridae).

A study of spermatozoa in the isthmus of the oviduct and of the surrounding epithelial cells in the dasyurid marsupial, Sminthopsis crassicaudata, was carried out. At least 10% of the ejaculated spermatozoa probably populate the isthmus region, where many come to reside in crypts until around the time of ovulation. Ultrastructural observations of spermatozoa in this region indicated that they had intact acrosomes and were identical in their morphology with those in the cauda epididymidis. After ovulation spermatozoa rapidly disappeared, some of which may be phagocytosed by the cells lining the crypts. These epithelial cells were also found to have many large, electron-dense granules at the time of sperm storage, but the contents did not appear to be released until the zygotes passed along the tract. The secretory activity of these cells may thus relate more to the production of the shell membrane that comes to surround the zygote than to the cells performing a nutritive or protective function for the spermatozoa during their period of storage within the female reproductive tract.

Animals↗

PubMed1989

Structure of the zona pellucida and cumulus oophorus in three species of native Australian rodents.

The sperm head of many Australian hydromyine rodents has three curved hooks projecting from its anterior margin; the structure of the hooks has been characterized, but their function is unknown. In this study, we have investigated whether the hooks might have evolved to assist sperm penetration through more formidable egg vestments, particularly the zona pellucida. Cumulus-oocyte complexes were obtained from two species that possess a three-hooked sperm head (Pseudomys australis and P. nanus) and one species that does not (Notomys alexis) and examined by light and electron microscopy. After fixation in the presence of ruthenium red, the zona pellucida was found to consist of a fibrillar meshwork, but there were no interspecific structural differences. A corona radiata was absent, and the cumulus extracellular matrix was composed of filaments and electron-dense granules in each species. Measurements of the zona thickness in freshly ovulated, unfixed oocytes revealed that it was thinnest (7.8 microns) in P. australis, which has a three-hooked sperm head, and thickest (11.4 microns) in N. alexis, the species in which the ventral hooks are absent. Hence, no correlation was found between the thickness of the zona pellucida or the structure of the cumulus-oocyte complex, and the presence of three hooks on the sperm head. We conclude, therefore, that it is unlikely that the evolution of the three-hooked sperm head is an adaptation for penetration of increased barriers around the oocyte.

Animals↗